Process for expression and production of recombinant protein hybrid protein (p24/p17) derived from human immunodeficiency viruses (HIV-1)

ABSTRACT

The present invention describes recombinant p24/p17 hybrid protein derived from the human immunodeficiency virus, their corresponding encoding recombinant DNA molecule and the process of production of the recombinant protein produced through genetic engineering techniques, to be used in diagnosis, vaccination or in research.

TECHNICAL FIELD OF THE INVENTION

The present invention refers to the general field of the technology ofthe DNA recombinant proteins, for the production of a recombinant hybridprotein (p24/p17) derived from human immunodeficiency virus (HIV-1) tobe used in diagnosis of acquired immunodeficiency disease virus (AIDS),vaccination, antibody production or in research.

BACKGROUND TO THE INVENTION

The epidemic provoked by the human immunodeficiency virus(HIV), todayglobal, continues without bamers, and has as a consequence the syndromeof the acquired immunodeficiency(AlDS), recognized 12 years ago. Thestudies of the World Organization of Health estimated that more than 18million people are infected and by the year 2000, 40 to 100 millionpeople worldwide would have been infected by HIV-1 or HIV-2, from which10 million will be children.

The hope for an effective therapy and, mainly, the prevention resides inthe acquisition of new information on the virus, the mechanisms of thepathogenesis and in the search of experimental models (COOPER. Immunol.4: 461, 1992). The current knowledge is that blocking the disseminationwill be very important and therefore the early detection is needed forthe treatment with the new drugs.

The virus concentration can greatly influenced the virus transmission ina body fluid. In the beginning of the epidemic the major routes of AIDStransmission were sexual contact and through transfusion withcontaminated blood (Levis in: HIV and pathogenesis of AIDS p.26,1989).The syndrome was initially described in homosexual and bisexual men andin intravenous drug users (Mansur et al.,N. Engl. J. Med.305:1431,1989,but its occurrence from heterosexual activity were soon recognized(Harris, C et al.,N.Engl. J. Med.308:1181,1983). Today the principalmeans of transmission are the sexual, maternal-child, drug users andstill blood in undeveloped countries. All of them can be explained to agreat extent by the relative concentration of HIV in various bodyfluids.

HIV entry in the body and developed acute infection as observed by othervirus. The HIV pathogenesis reflects several properties of the virus andthe host immune response. AIDS final outcome is the differentialexpression of those major components of HIV infection

The first step of HIV infection is the interaction between the majorlymphocyte receptor CD4 molecule, a member of immunoglobulin superfamilywith the envelope gp120 protein. According to some reports described gp120 is displaced, leading to the uncovering of domain on the envelopegp41 needed for virus cell fusion. Once in the cytoplasm severalintracellular events take place ending with integration of a proviralform into cellular genome.

Besides entering cells via the direct interaction of the virus envelopewith cell surface receptors, HIV can infect cells by other mechanisms.For example, during the course of studies on the humoral response toHIV-1 (infection, the phenomenon of antibody dependent enhancement(ADE), of HIV infection was found to occur (Homsy, J. et al. Science244: 1357, 1989; Homsy, J. et al. Lancet i: 1285, 1988). The transfer ofthe virus into a cell through the complement or Fc receptor involves thebinding of the Fab portion of nonneutralizing antibodies to the virussurface (Levy in: HIV and the pathogenesis.p.53,1994)

Clinical manifestations of acute HIV infection were recognized in thevery early studies, and have been described in various articles(Thindall and Cooper, AIDS:5:1, 1991; NIV, MT et al. J. Infect. Dis.168:1490, 1993). A newly infected host present within 1 to 3 weekssymptoms as headache, pain, muscle aches, sore throat, fever, swollenlymph nodes, a non pruritic macular erythematous rash involving thetrunk and later the extremities.

It is estimated that 50 to 70% of patients primary infected with HIVwill developed a syndrome of acute mononucleosis-like illness. Thisperiod is associated with high viremia levels, and the immune humoralresponse against the virus is detected between one week and three months(DAAR et al. New Engl. J. Med 324: 961,1991).

This specific immunity that initiates in this period is associated to adramatic decline of the viremia, but the level of this immunityinadequate to suppress the viral multiplication. The expression of thevirus persists in the lymph nodes, even when the presence of the virusin the plasma is difficult to be detected, and the mRNA is notdetectable in the mononuclear cells of blood stream (MICHAEL et al. J.Virol. 66: 310-316, 1992).

HIV is classified, based on its morphology, genomic organization andpathogenic properties, as a member of the sub-family lentivirus of thefamily Retrovindae.

HIV-1 and HIV-2 as a lentivirus have the characteristic cone-shaped corecomposed of the viral p24 GAG protein. Inside this capsid are twoidentical RNA strands with the reverse transcriptase (RNA dependent DNApolymerase) and the nucleocapsid proteins (p9, p6) are closelyassociated. The inner portion of the viral membrane is surrounded by amyristylated p17 core protein (GAG) that provides the matrix (MA) forthe viral structure which is important for virion integrity (McCune, J.M. et al. Cell 53: 55, 1988; Shulz, T. F. et al. AIDS Res. Human Retrov8:1584, 1992). The virus surface is made by envelope glycoproteinsderived from a precursors of Mw 160.000 which is inserted inside thecell into a gp120 and a gp41 transmembrane protein (TM). The centralregion of TM protein binds to the external viral gp120 in a non covalentligation at two hydrophobic regions in the amino and carboxi termini ofgp120 (Helseth, E. et al. J.Virol. 65:2119-2123, 1991.

Two other genes tat and rev are positive regulators for the replicationof HIV, besides other proteins with accessory function, as the vpu, vif,vpr and nef (ROSEN, TIG 7: 9-14. 1991.).

One of the most notable properties of the genoma of the HIV-1 is itsgenetic variation (DESAI et al Proc. Natl. Acad. Sci. 83: 8380, 1986).The diversity can be important for many aspects of the biology of thevirus, among them the tissue and cell specificity, clinical-pathologicalpicture of the disease, geographical and temporary virus distribution,difference in the susceptibility of immune response, virulence and,especially, development of a vaccine of wide crossed reactivity. Themistake range esteemed for the variation of HIV is of a substitution in10⁴ synthesized nucleotides. Besides the substitution, deletions andinserts, whose frequency is more difficult of being evaluated canhappen.

The gene env shows, along its structure, variable (V) and constant (C)regions. The principal neutralizing domain of the HIV-1 is placed in thethird variable region (it raises V3) of the envelop glycoprotein(gp120). The loop V3 is an important neutralization epitop for, viraltropism and syncytium formation. In the V3 loop is the neutralyzingepitop for type-specific antibodies.

The laboratory diagnosis of any infectious agent is an important aspectfor the control of infectious diseases. The precise diagnosis winsimportance in the blood derivatives, whose use depends on the capacityof the tests in the detection of infectious agents' or its antigens.During the last decade, the technological progresses developed precisetests for the diagnosis of AIDS. Most of the researches concentratedefforts for the development of tests to be used in development countrieswere are the economic limitation. In those countries the HIV detectionis not a routine.

The HIV-1 diagnosis is made with the indirect detection of the presenceof the virus, indicated by the patient's immune response, evidenced bythe presence of specific antibodies against the HIV-1 or the detectionof the virus or its components.

The direct methods identify the virus multiplication in culture,detection of virus or antigens in immunoassays (ELISA), molecularhybridization or amplification of nucleic acids (PCR). However, some ofthese tests demand qualified personnel and equipped laboratories, whathinders its widespread use. An exception is the test of ELISA for theantigen p24, that can be detected in patients before the detection ofantibodies, even so, in some cases, it is only detected in lateinfections and in some patient were it seems to be transient. Thus, thenegative result of tests for HIV-1 antigens is not informative and itdoesn't necessarily reflect a not infected individual (BYLUND et al.Clin. Lab. Med. 12: 305, 1992)

THE indirect methods determine the detection of antibodies. However,these antibodies are detected a time after infection from six weeks tosix months.

Other indirect tests are those that not measure specific immune responsebut some proteins as β-2-microglobulina and neopterine, that indicatethe activation of the immune response.

The detection of HIV-1 antibodies is the most used and efficient methodto demonstrate the patient's contact with the virus or to verify thecontamination of blood samples. In 1988, laboratories linked to theprogram Performance Evaluation Program of the Center of Control ofDiseases (CDC, Atlanta, USA), evaluated around 32 million tests usingantibody anti-HIV. A test of antibody anti-HIV is considered positive,when a sequence of tests, beginning with repeated ELISA and includingadditional, more more specific test, as the Western blot, they areconsistently positive (CDC, MMWR ,1987). (BYLUND et al. Clin. Lab. Med.12: 305, 1992).

The ELISA test is most used for detection of the HIV-1 because of thelow cost, easy standardization and execution, Initially, it waslicensed, in 1985, to test blood donors and blood products, being theiruse expanded to determine antibodies anti-HIV-1 in populations (Weiss etal. JAMA 253: 221, 1985). Several studies described the high sensibilityof this test from 93% to 100% (BYLUND et al. Clin. Lab. Med. 12: 305,1992).). Several kits of ELISA were licensed by the “Food and DrugAdministration ” (FDA), in United States. Most of the tests usedinactivated and purified lysates of T cell lineage, H-9, as source ofantigen, that is rich in p24 and p17 antigens, with some loss of gp160,gp120 and p41 during the preparation. The contamination of thepreparations of antigens with cell debris can originate the falsepositive result, due to cross reactions. These problems were nowresolved with the use of recombinant antigens or synthetic peptides inthe tests of ELISA, that constitute the last generation of tests todetect the antibody anti-HIV.

The recombinant proteins produced in bacterias and yeasts has been usedas antigen for different types of tests, like ELISA,radioimmunoprecipitation, latex agglutination and Western blot. Thesensibility and the specificity of these methods are excellent (99 to100%), and they can detect the serum conversion earlier than ELISA thatuses antigens of total virus.

The Western blot is the most used complementary test for the detectionof specific antibody anti-HIV-1. In comparison to ELISA the Western blotis of higher cost and it requests technical personnel specialized due tosubjective interpretation, because a universal approach doesn't existfor the interpretation of positive cases.

The bands of gp120 and gp4l don't have a good resolution, because theseare glycosilated proteins of the envelope that migrates slowly in thegel, being considered an only band, for the interpretation of theresults. A negative test is doesn't present any band, however thepresence of an only band doesn't fill the requirements for a positivetest and it is considered uncertain. This approach perhaps is not idealto be used patients of high risk, or for patients with suggestivesymptoms of HIV infection, especially if the band of p24 is detected(KLEINMAN. Arch. Pathol. Lab. Med., v.114, p.298, 1990.).

The false positive reactions can be observed in the ELISA test in theearly and late phases of the patient's infection by HIV-1.False-positive results were described in patients withhiperbilirubinemie, disorders of the connective tissue, polyclonalgamopatias, besides in healthy individuals, as a result of a not veryunderstood cross-reaction. However, it was verified that, in apopulation of low risk, the index of false-positive reactions of thetests of ELISA and Western blot combined was smaller than 10⁻⁵ (BURKE etal., New Engl. J. Med. 319: 961. 1988).

Thus, there is need of a system with high sensibility for the detectionof the virus, its components or antibodies from infected individuals' Animportant fact is it that the synthetic peptides and recombinantproteins are superior to the antigens from cell lysates. Thus, throughthe genetic engineering, it is possible to construct hybrid proteinsthat combine antigenic characteristic of more than one viral component.

It is object of the present invention to describe the recombinant hybridp24/p17 protein of HIV-1, their corresponding encoding recombinant DNAmolecule and the process of production of the recombinant hybrid p24/p17protein of HIV-1 produced through techniques of genetic engineering, tobe used for diagnosis, vaccination or in research.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other objects, features and many attendant advantages of theinvention will be better understood upon a reading of the followingdetailed description when considered in connection with the accompanyingdrawings wherein:

FIG. 1 shows the vector pDS56 used for the expression of the recombinanthybrid p24/p17 protein of HIV-1

FIG. 2 shows the amino acid sequence of the recombinant hybrid p24/p17protein of HIV-1

FIG. 3 shows the hydrofilicity profile from the recombinant hybridp24/p17 protein of HIV-1

FIG. 4 shows a PAGE with the purified recombinant hybrid p24/p17 proteinof HIV-1

DETAILED DESCRIPTION OF THE INVENTION

The methodology used for the production of the recombinant hybridp24/p17 protein of HIV-1consists of the cloning and expression, inmicroorganisms, of the DNA corresponding to the gene that codesrecombinant hybrid p24/p17 protein of HIV-1 using the methodology of thegenetic engineering.

In order to better understand this invention the following examples, forillustrative purposes only, are described. The examples illustrate thepresent invention and are not intended to limit it in spirit or scope.The process can be understood better through the following descriptionin consonance with the examples.

EXAMPLE 1

Amplification of the DNA (1)

The amplification of the DNA (1) derived from the proviral DNA, orstarting from the vector that contains the cloned DNA of the gene forP-24/p17 hybrid protein was developed using specific oligonucleotides 5′GGATCCCCGCTGACATGGAGCAAGGCG3 (SEQ ID NO. 1) and 5′CGCGAAGCTTCAGGCTCCATCTGTC3′ (SEQ ID NO. 2). Those oligonucleotides weredrawn to amplify, through polymerase chain reaction (PCR), the DNAregion that encodes the corresponding fragment of the P-24/p17 hybridprotein. The primers also contains the sites for the restriction enzymesBamH-1 and Hind III.

The PCR reaction was performed by using Taq polymerase buffer (50 mMKCI, 100 mM Tris-HCI pH 9.0-9.5, 1.5-2.5 mM MgCI₂ and 1-2% tritonX-100), 0.1-1 U of Taq polymerase (Promega ,E.U.A., Cat. no. M186A),0.5-1.5 mM MgCI₂, 20-50 mM of each nucleotide (dATP,dCTP,dGTP,dTTP)10-30 umoles of each primer, and 0.01a 0.1 μg cDNA and H₂O q.s.p. 50-100μI. The reaction was performed in 1-2 cycles at 94-96° C./1-2 min; 53 to55° C. 1-2 min.; 70-72° C./1-2 min; 30 cycles at 94-96° C./1 to 2min;36-38° C./1-2min; 70-72° C./1-2 min and more 1 cycle to 94-96° C./1-2min; 36-38° C./1 to 2 min; 70-72° C./10-15 min.

The PCR product was fractionated by electrophoresis in 1.5-2.0% agarosegel before purification of amplified fragment band was cutted out thegel. The fragment was purified by adding 2-3 times v/v of Nal solution(Nal 8M+0.022 M DTT) and sodium phosphate buffer (1M pH 6.0-6.5) andincubated for 5-10 min. at 50-56° C. Glass beads were added to thesuspension, mixed , incubated 1-5 min at room temperature andcentrifuged 10-30 seconds . The pellet were washed with ethanolbuffer(75% of ethanol, 0.01 M Tris-HCI, pH 7.0-7.6, 0.01 M EDTA, pH8.0-8.5). The DNA was eluted from the glass spheres with buffer (Tris pH7.0-7.4 10 mM, 1-3 mM EDTA) at 50-56° C. for 1-5 min.

EXAMPLE 2

Cloning (2)

The PCR product was digested with enzyme Hind III with 10-20 U of HindIII (Biolabs, England) plus 3-5 I buffer (Promega, EUA) in 30-50 μlvolume of H₂O. The reactions were incubated at 37° C. for 2-4 h. Afterthis time 10-20 U of Bam HI (Biolabs, England) plus 5-10 μI of react IIIbuffer (BRL, USA) were added to a final 50-100 μI volume of H₂O dd andit was incubated at 37° C. for 2-4 h. For cloning of the PCR productinto plasmid PDS-56 (FIG. 1), the vector was digested with 10-20 U ofenzyme Hind III(Promega, USA), 2-5 μI buffer I B (Promega,E.U.A.) in20-50 μI final volume of H₂O , and incubation at 37° C. for 2-4 h. Tothe reaction was added 10-20 U of the enzyme Bam Hi (Promega, USA), 5-10μI of react III (BRL, E.U.A), in 50-100 μfinal volume of H₂O , andincubation at 37° C. for 2-4 h. After digestion the DNA was fractionatedby electrophoresis in a 1-2% TAE-agarose gel and bands purified asalready described.

The ligation reaction was performed by adding 20-50 ng of the DNAfragment insert, 5-15 ng of the vector DNA, plus 0.5-2.0 U of T4 ligase(Promega, USA), 5mM ATP (Promega,E.U.A.), ligationbuffer(Promega,E.U.A.), H₂O dd qsp 15 μI, with incubation at 14-16° C.(BOD, FANEN, Brazil) for 12-18 h.

EXAMPLE 3

Transformation (3)

The bacterial transformation was done with Escherichia coli by addingthe ligation reaction completed to 40-60 μl volume buffer (Tris 10 mM pH7.2-7.4, EDTA 1 mM) to 100 μl of competent bacteria suspension. Thetubes were slightly rotated and immediately incubated on ice bath for20-40 min. After that, they were submitted to a thermal shock at 40-42°C. for 1-3 min. and kept on ice bath for further 20-40 seconds. LBmedium (Bacto triptona 1% p/v, extract of yeast 0.5% p/v, NaCl 171 mM)without antibiotic was added at double volume and incubated at 37° C.for 1-2h. The bacteria were pelleted, homogenized in LB and inoculatedin Petri dish plates with LB agar (agar 1.5% p/v, yeast extract 0.5%p/v, triptone 0.1% p/v, NaCl 0.5% p/v pH 7.2-7.5) with 50-200 pg/mlampicillin and 20-100 pg/ml kanamycin. The plates were incubated at 37°C. for 15-24 h. For the selection of the positive clones they were grownin LB with 50-200 pg/ml ampicillin and 20-100 pg/ml kanamycin at 37° C.under agitation for 15-20 h. After incubation a PCR using specificprimers of the vector (for amplification of the area corresponding toinsert) being the primer (sense) 5′-TTCATTAAAGAGGAGAAATT-3′(SEQ ID NO.3) and primer (anti-sense) 5′-CTATCAACAGGAGTCCAAGC-3′(SEQ ID NO. 4). Thereaction was made with Taq. polymerase buffer10X (KCl 500 mM, Tris-HCl100 mM pH 9.0-9.5, MgCl₂ 15-25 mM and triton X-100 1-2%), 0.5-1.0 U ofTaq polymerase (Promega, USA), 0.5-1.5 mM MgCl₂, 20-50 mM of eachnucleotide (dATP, dCTP, dGTP, dTTP), 10-30 pmoles of each primer, 0.5-1μl of bacterial suspension and H₂ Odd sterile qsp 20-40 μl.

The reaction was processed with 1-3 cycles of 94-96° C./5 min., 50-55°C./1-2 min., 70-72° C./1-2 min., 30 cycles of 94-96° C./30-45 seg.,45-50° C./30-45 seg., 70-72° C./30-45 seg. and 1 cycle of 94-96° C./1-2min., 45-50° C./1-2 min., 70-72° C./10-15 min. The of this reaction wasfractionated through 1-2%. agarose gel electrophoresis.

EXAMPLE 4

Sequencing (4)

The positive clones were sequenced to confirm the sequence of FIG. 2 andpresents the hydrofilicity profile as showed in FIG. 3.

EXAMPLE 5

Protein production (5)

The positive clones were used for production of protein and they weregrown in LB medium with 50-200 μg/ml ampicillin, 50-200 of Kanamycinμg/ml and incubated at 37° C. under agitation until the optical density(OD 600 nm) of 0.5-0.7. Then, for the induction of the protein,IPTG(Isopropyl-□-D-thiogalactpyranoside) to 0.2-0.4 M was added andincubated for 3-5 h. The bacteria were centrifuged, the supernatant wasdiscarded and the pellet homogenized in buffer A (guanidine-HCI 5-6 M,sodium phosphate 0.1-0.2 M, Tris 0.01-0.02 M pH 7.8-8.0) with agitationfor 1-2 h. A polyacrylamide gel shows the expression in the bacteria.(FIG. 4)

EXAMPLE 6

Protein purification (6)

After the centrifugation the supernatant was applied to a column withNi-NTA (nickel chelate) resin. For purification of the protein thecolumn was washed sequentially with buffer A, buffer B (Urea 7-8 M,phosphate of sodium 0,1-0,2 M, Tris 0.01-0.02 M pH 7.8-8.0) and withbuffer C (Urea 7-8 M, phosphate of sodium 0.1-0.2 M, Tris 0.01-0.02 M pH7.0-7.2). The protein was eluted with buffer D (Urea 7-8 M, sodiumphosphate 0.1-0.2 M, Tris 0.01-0.02 M pH 5.0-5.2) and sequentially withurea 7-8 M, phosphate of sodium 0.1-0.2 M, Tris 0.01-0.02 M pH 40-4.2.Fractions were collected and 50 μI of each fraction was diluted v/v insample buffer, heated for 10 min. and submitted to electrophoresis inpolyacrylamide gel (SDS-PAGE).

While the present invention has been described in connection withexamples, it will be understood that modifications and variationsapparent to those ordinary skill in the art are within the scope of thepresent invention.

5 1 27 DNA Human immunodeficiency virus 1 ggatccccgc tgacatggag caaggcg27 2 25 DNA Human immunodeficiency virus 2 cgcgaagctt caggctccat ctgtc25 3 20 DNA Artificial Sequence Description of Artificial Sequence PCT,primer of the vector 3 ttcattaaag aggagaaatt 20 4 20 DNA ArtificialSequence Description of Artificial Sequence PCT, primer of the vector 4ctatcaacag gagtccaagc 20 5 199 PRT Human immunodeficiency virus 5 GluAla Leu Asp Lys Ile Glu Glu Glu Gln Asn Lys Ser Lys Lys Lys 1 5 10 15Ala Gln Gln Ala Ala Ala Asp Thr Gly His Ser Ser Gln Val Ser Gln 20 25 30Asn Tyr Pro Ile Val Gln Asn Ile Gln Gly Gln Met Val His Gln Ala 35 40 45Ile Ser Pro Arg Thr Leu Asn Ala Trp Val Lys Val Val Glu Glu Lys 50 55 60Ala Phe Ser Pro Glu Val Ile Pro Met Phe Ser Ala Leu Ser Glu Gly 65 70 7580 Ala Thr Pro Gln Asp Leu Asn Thr Met Leu Asn Thr Val Gly Gly His 85 9095 Gln Ala Ala Met Gln Met Leu Lys Glu Thr Ile Asn Glu Glu Ala Ala 100105 110 Glu Trp Asp Arg Val His Pro Val His Ala Gly Pro Ile Ala Pro Gly115 120 125 Gln Met Arg Glu Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr SerThr 130 135 140 Leu Gln Glu Gln Ile Gly Trp Met Thr Asn Asn Pro Pro IlePro Val 145 150 155 160 Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu Gly LeuAsn Lys Ile Val 165 170 175 Arg Met Tyr Ser Pro Thr Ser Ile Leu Asp IleArg Gln Gly Pro Lys 180 185 190 Glu Pro Phe Arg Asp Tyr Val 195

What is claimed is:
 1. A recombinant hybrid p24/p17 protein of HIV-1consisting of SEQ ID NO. 5.